Assay Overview: The assay detects intracellular trypanosomes that are replicating inside host cells. NIH3T3 cells are trypsinized, counted, and plated in 384-well tissue culture plates. After plating the NIH3T3 cells, compounds are added to the wells. T. cruzi that are genetically modified to express b-galactosidase are then harvested and active Trypomastigotes form are counted and co-cultured with the NIH3T3 cells. After 90 hours of co-culture, all cells in the well are lysed and b-galactosidase production is detected using a luminescent reporter system (GalScreen). ..more

Assay Overview: The assay detects intracellular trypanosomes that are replicating inside host cells. NIH3T3 cells are trypsinized, counted, and plated in 384-well tissue culture plates. After plating the NIH3T3 cells, compounds are added to the wells. T. cruzi that are genetically modified to express b-galactosidase are then harvested and active Trypomastigotes form are counted and co-cultured with the NIH3T3 cells. After 90 hours of co-culture, all cells in the well are lysed and b-galactosidase production is detected using a luminescent reporter system (GalScreen).

Expected Outcome: Compounds significantly suppressing luminescence, and therefore beta-galactosidases expression will be identified as hits in the screen. Compounds that inhibit luminescence activity may kill T. cruzi, inhibit T. cruzi invasion or inhibit development of the parasite within the host cell. Compounds that are toxic to the host cell will be excluded in secondary assays.

PROTOCOL HTS ASSAY in 384-well plate TRYPANOSOMA CRUZICells and parasite:LLC-MK2 cells (rhesus monkey kidney epithelial cell line)NIH/3T3 cells (mouse embryonic fibroblast cell line)Trypanosoma cruzi expressing beta-galactosidase (Tc-beta-gal: Tulahuen strain, clone C4 from: Buckner et al., 1996, Antimicrobial agents and chemotherapy, 40:11, pp 2592-2597.)Reagents, materials, solutions and culture media:Reagents:DMEM with Phenol Red, high glucose, with L-glutamine and Sodium pyruvate (Cellgro, cat number: 10-013-CM)PSG or Penicillin-streptomycin-L-glutamine (Gibco-Invitrogen, cat number: 10378-016)FBS-Heat inactivated fetal bovine serum (Gibco-invitrogen, cat number: 16140-089)0.25% Trypsin-EDTA 1X (Gibco-Invitrogen, cat number: 25200-072)Sterile PBS (Phosphate Buffer Saline) 1XNP40 (Nonidet P-40, now called Igepal CA 360, Fluka, cat number: 56741)GalScreen, Buffer B (Applied Biosystems, cat number: T1031)Materials:T175, T225 culture flasks with vented caps (BD Falcon, cat number: 353028)Corning Hyperflasks (Corning, cat number: 10024)Culture medium:For cell propagation: 90% DMEM+Phenol red, 10% FBS, 1% PSG. Mix and filter through 0.2 microns membrane. Keep at 4#C. Warm up to 37#C in water bath before use.For Tc culture and assays:98% DMEM+Phenol red, 2% FBS, 1% PSG. Mix and filter through 0.2 microns membrane. Keep at 4#C. Warm up to 37#C in water bath before use.Solutions:GalScreen + 0.05% NP40. Using GalScreen base kit (Applied Biosystems), mix Buffer B (Applied Biosystems, T2361) with 1:25 substrate (Applied Biosystems, T2359) with 1:400 dilution of 20%NP40. Parasite and cell cultureCell culture : NIH/3T3Notes:NIH/3T3 cells are cultivated in DMEM supplemented with 10% FBS and 1% PSG in T175 or Corning Hyperflasks . Conditions can be adapted to other culture plate sizes. Cells are usually passaged Protocol:Aspirate medium. Rinse cells with 10 ml PBS/plate. Aspirate PBS. Add 4 ml of pre-warmed trypsin-EDTA, swirl the dish to make sure the trypsin covers all the cells. Leave the dish a few minutes at room temperature (usually not more than 5). Check that the cells are detaching. Add 21 ml of medium, pipet up and down to detach all the cells. Take 75 ul of media and add to Cellometer cassette (Nexcelom Biosciences). Using the Cellometer Auto T4 (Nexcelom Biosciences), focus so cell bodies have dark edges and clear/white centers. Select NIH3T3 preset menu, dilution 1x, display image, and count. Using this number dilute in either 2% FBS/DMEM for assays or 10% FBS/DMEM for propagationCell culture : LLC-MK2Notes:LLC-MK2 cells are cultivated in DMEM +Phenol red supplemented with 10% FBS and 1% PSG in T175 flasks in 35 ml total of medium. Conditions can be adapted to other culture plate sizes. Cells are usually passaged twice a week at 1:4 to 1:8 ratios. Protocol:Aspirate medium. Rinse cells with 10 ml PBS/plate. Aspirate PBS. Add 5 ml of pre-warmed trypsin-EDTA, swirl the dish to make sure the trypsin covers all the cells.Place the dishes back in the incubator for 5 min. Check that the cells are detaching. Add 20 ml of medium, pipet up and down to detach all the cells. Dilute into 2% FBS/DMEM for assay or 10% FBS/DMEM for propagation.Parasite culture: Trypanosoma cruzi beta-gal (Tc)Notes:Tc-β-gal are cultivated in DMEM +Phenol red supplemented with 2% FBS and 1% PSG in T175 flasks with vented caps in 35 ml total of medium.Protocol:The day before infection (or at least 2-3 hours before), plate 3 Million LLC-MK2 cells/T175 in DMEM+10% FBSEither thaw Tc in from liquid nitrogen stock into 50 ml 2% FBS/DMEM or remove the media from a propagating LLC-MK2 co-culture, in other words, harvesting the medium containing the free Tc in 50 ml falcon tubes.Spin 10 min at 2200 rpmAspirate the supernatant until 15 ml is leftPlace back the tubes in the incubator delicately (so as not to disturb the pellet)Incubate minimum 3 hours at 37#C.Take supernatant to fresh tube.To count: Mix 75 ul Tc with 25 ul 16% PFA, mix, put 75 ul Cellometer cassette and count using the Cellometer Auto M10 (Nexcelom Biosciences). Wait for 2-5 min to let the Tc settle, press display image, focus, and count. Note, if there are numerous bubbles or low numbers of Tc, the counter will be inaccurate. It is critical to check every screen to see that it has properly counted. Often, there is ~ 10-20% over counting. Alternatively, TC can be counted a hemacytometer using the smaller/inner grids.Leave Tc incubating in 50 ml conicals until ready to dilute for experiment or inoculate new LLC-MK2 culture.Aspirate LLC-MK2 media (used 10% for plating) and replace with 2% FBS/DMEM.Plate 17-35 Million on Tc on fresh LLC-MK2Change medium after 2 days (Use 2% FBS/DMEM).Harvest Tc trypomastigotes on Day 5, 6 and/7. Growth inhibition assay for HTS in 384 well plates:Notes:Final volume per well for cells+parasites+compound is 50 ul.Final volume after adding substrate is ~65 ml. (evaporation of 50ul to 35 ul after 90 hrs, and then 30ul GalScreen addition)The assay is performed in DMEM+ 2% FBS and 1% PSG . Experiment set up is usually done ~12pm. Assay has to be incubated 90 hrs or a bit more.Protocol:warm up medium 2% FBS/DMEMharvest parasites in 50 ml tubes (1 flasks per tube)spin 10 min at 2200 rpmAspirate media ~ 15 ml and place them on a rack in the incubator to let trypomastigotes swim out of the pellet for 3-5 hours.In the meantime, trypsinize NIH/3T3 cells as described in cell culture protocolWhen NIH3T3 are detached, harvest them in DMEM- 2% FBS and 1% PSG and count using the NIH3T3 program using Cellometer Auto T4 (Nexcelom Biosciences). Dilute cells to 166,667 cells /ml and add to flask.plate 5,000 cells/30ul per well using a Thermo MultiDrop Combi liquid dispenser and a sterilized standard sized dispensing cassette adding at a fast speed in a tissue culture hood.Put back in incubator for 3 hours to allow cells to attach.Count Tc as from protocol above.Dilute to 0.250 million /ml and transfer a 2 liter flask.Pin 50 nl compounds/DMSO to each wellAdd shortly after 20 ul/well of parasites (5000 Tc) using a Thermo MultiDrop Combi liquid dispenser and a sterilized standard sized dispensing cassette adding at slow speed.Incubate for 4 days (or minimum 90 hours).On day of substrate addition, prepare GalScreen with 0.05% NP40 (Buffer B with 1:25 dilution substrate with 1:400 dilution of 20% NP40).Add 30 ul per well of 384 well plate using a Thermo MultiDrop Combi liquid dispenser and a sterilized standard sized dispensing cassette adding at a fast speed.Incubate for 60 minutesRead using the ultrasensitive luminescence program on the Envision (Perkin Elmer).

HTS Data Analysis:Negative control wells (DMSO) were included on every plate.The PubChem_Activity_Score was derived using the follow procedure:1. An activity score for each well was derived using Genedata Screener Assay Analyzer (v6.0.1). The value of each well was normalized using the "Neutral Contols" method. When using this method, a background-subtracted value is derived by subtracting the median value of the negative control wells on each plate from the value of each well on that plate. This background-subtracted value is then divided by the median of the negative controls on the plate and multiplied by -100 to derive the normalized value.2. Systematic plate effects were then corrected for each run by using the "Runwise Pattern (Additive)" correction method of Genedata Screener Assay Analyzer (v.6.0.1).3. The final PubChem_Activity_Score represents the mean of all valid replicate activity scores.

The PubChem_Activity_Outcome was assigned as described below:Activity_Outcome = 1 (inactive)PubChem_Activity_Score >-55 and less than half of all replicates have PubChem_Activity_Score <=-55Activity_Outcome = 2 (active)PubChem_Activity_Score <=-55Activity_Outcome = 3 (inconclusive)PubChem_Activity_Score > -55, but at least half of all replicates have PubChem_Activity_Score <=-55.Activity_Outcome = 4NA